Modulation of Hepatic ABC Transporters by Eruca Vesicaria Intake: Potential Diet-Drug Interactions T

Food and Chemical Toxicology 133 (2019) 110797

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Food and Chemical Toxicology

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Modulation of hepatic ABC transporters by Eruca vesicaria intake: Potential diet-drug interactions T

Martín I. Romaa,1, Victoria E. Schiariti Lampropulosb,1, Iván Ayllón-Cabrerab, ∗ Ana N. Salazar Sanabriaa, Marcela M. López Nigrob, Roxana N. Peronia,2, , Marta A. Carballob,2 a Universidad de Buenos Aires, Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas, Instituto de Investigaciones Farmacológicas (ININFA), Facultad de Farmacia y Bioquímica, Junín 956, C1113AAD, Buenos Aires, Argentina b Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Fisiopatología y Bioquímica Clínica (INFIBIOC) - Depto. de Bioquímica Clínica, CIGETOX (Citogenética Humana y Genética Toxicológica), Junín 956, C1113AAD, Buenos Aires, Argentina

ARTICLE INFO ABSTRACT

Keywords: The aim of this work was to evaluate whether oral administration of Eruca vesicaria, a species of rocket cultivated Brassicaceae in Argentina, could modify cyclophosphamide (CP)-induced genotoxicity through modulation of hepatic ABC ABC transporters transporters. Daily oral administration of E. vesicaria fresh leaves juice (1.0, 1.4 and 2.0 g/kg) for 14 days did not fi Cell detoxi cation alter genotoxicity biomarkers —alkaline comet assay and micronucleus test —in neither male nor female mice. DNA damage protection Instead, repeated intake of this cruciferous decreased CP-induced DNA damage dose-dependently and it caused Cancer hepatic overexpression of P-glycoprotein (P-gp; 1.4 and 2.0 g/kg) and multidrug resistance protein 2 (MRP2; 2.0 g/kg), but not breast cancer resistance protein (Bcrp). The antigenotoxic effect of E. vesicaria was prevented by 50 mg/kg verapamil (P-gp inhibitor) or 10 mg/kg indomethacin (MRP2 inhibitor). In turn, CP-induced cytotoxicity (10 mM, 24 h) on human hepatoma cells (HepG2/C3A) was significantly reduced by preincubation with E. vesicaria (1.4 mg/ml; 48 h); this effect was absent when CP was coincubated with 35 μM verapamil, 80 μM indomethacin or 10 μM KO-143 (BCRP inhibitor). Altogether, these results allow us to demonstrate that repeated intake of E. vesicaria exhibited antigenotoxicity, at least in part, by induction of hepatic ABC transporters in vivo in mice as well as in vitro in human liver cells. This could account for other diet-drug interactions.

1. Introduction fractions rich in isothiocyanates and other glucosinolate-breakdown products from several Brassicaceae vegetables can cause DNA damage The widespread use of herbal medicines, nutraceuticals and dietary and clastogenicity in mammalian cells (Baasanjav-Gerber et al., 2011; natural products concomitantly with pharmacotherapy has raised con- Fimognari et al., 2012; Kassie et al., 1996; Kupke et al., 2016; Wiesner cerns on their possible interactions and, therefore, effect on drugs ef- et al., 2014). Both deleterious and beneficial effects of isolated com- ficacy. Herbs and dietary ingredients can alter transport or metabolic pounds of cruciferae were associated with tissue-specific modulation of processes, affecting drug disposition (Fugh-Berman, 2000; Izzo and cytochrome P450 and phase II conjugation systems in several in vitro Ernst, 2009). and in vivo models of cancer (Canistro et al., 2012; Higdon et al., 2007; The cultivated plant Eruca vesicaria—common name “rocket”—is Konsue and Ioannides, 2008; Paolini et al., 2003; Perocco et al., 2006; an edible cruciferous vegetable (Brassicaceae family) whose consump- Plate and Gallaher, 2006). tion has increased in the last ten years in Argentina. Several cruciferae However, the induction of hepatic enzymes caused by glucosino- extracts or their isolated phytochemicals, such as sulforaphane, erucin lates only partially reproduced the potent antigenotoxic effect of black and erysolin, have shown protective activity against DNA damage in- cabbage seeds or Eruca sativa leaf juice on human hepatoma cells ducers both in vitro and in vivo (Jin et al., 2009; Lamy et al., 2008; (HepG2) (Canistro et al., 2012; Lamy et al., 2008). This supports the Sharma et al., 2016). On the other hand, it has also been described that theory that several phytochemicals would act synergistically through

∗ Corresponding author. E-mail address: [email protected] (R.N. Peroni). 1 these authors contributed equally to this study. 2 these authors contributed equally to the direction of this study. https://doi.org/10.1016/j.fct.2019.110797 Received 21 March 2019; Received in revised form 2 August 2019; Accepted 28 August 2019 Available online 31 August 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved. M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797 different mechanisms of action to achieve the beneficial effect. 2.2. Preparation and phytochemical screening of Eruca vesicaria juice In this sense, phytochemicals found in cruciferous extracts and juices, including E. vesicaria, interact with ATP Binding Cassette (ABC) E. vesicaria leaves were obtained from certified organic gardens and transporters (Aszalos, 2008; Baiceanu et al., 2015; Dewanjee et al., processed according to Lopez Nigro et al. (López Nigro et al., 2018). 2017; Ebert et al., 2006; Miron et al., 2017; Telang et al., 2009). ABC Briefly, leaves were washed and dried and the stalks were removed transporters are involved in cell detoxification (Xu et al., 2005)by prior to weighting. The remaining plant material (310.6 g) was pro- expeling xenobiotics or endogenous waste products against concentra- cessed with a commercial juice maker, and the juice was afterwards tion gradients in an active ATP-dependent way (Efferth and Volm, centrifuged (14,000 rpm for 20 min at 4 °C). The supernatant (122 ml) 2017). P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistance pro- was clarified, filter-sterilized (0.22 μm) and diluted according to ex- tein 2 (MRP2/ABCC2 or its murine homologue Mrp2) and breast cancer perimental requirements. The juice was kept on ice and away from the resistance protein (BCRP/ABCG2 or its murine homologue Bcrp) are light throughout the entire procedure. Small aliquots were frozen at ABC transporters expressed in the canalicular membrane of hepato- −80 °C until use. cytes, which pour their substrates into the bile (Schinkel and Jonker, Subsequently, we performed a quantitative phytochemical 2003). Expression and activity of ABC transporters vary between in- screening of some of the main active components of the juice. For the dividuals due to sex, genetic polymorphisms, pathological conditions, assessment of total phenols, the Folin-Ciocalteu method was used xenobiotic exposure or dietary habits, leading to differences in bioa- (Makkar et al., 1993). Absorbance was recorded at 725 nm and gallic vailability of different drugs, toxins or food-derived carcinogens (Lu acid was used as a standard for the calibration curve. Total flavonoids and Klaassen, 2007; Patel et al., 2016; Suzuki et al., 2006). Previous were determined by adding aluminium chloride and sodium acetate to studies from our work group demonstrated that watercress and wild aliquots of the juice and recording the absorbance at 430 nm after room rocket (Diplotaxis tenuifolia) exhibit a protective effect against genotoxic temperature incubation (Maksimović et al., 2005). A rutin calibration damage induced by the alkylating agent cyclophosphamide (CP) curve was used for quantification. Lastly, the modified Dao and (Casanova et al., 2013; Casanova and Carballo, 2011; López Nigro et al., Friedman method was performed for the measurement of hydro- 2018). CP is an indirect mutagen metabolized by hepatic cytochrome xycinnamic acids (Dao and Friedman, 1992). Aliquots of 50 μl of the P450 system to several metabolites including the active 4-hydro- juice were diluted in 2 ml ethanol and absorbance was measured at xycyclophosphamide (OH-CP) that circulates in blood and enters cells 328 nm, using a chlorogenic acid calibration curve for quantification. as the tautomer aldophosphamide (Fleming, 1997). In turn, aldophosphamide is transformed into phosphoramide mustard and acrolein, 2.3. Animals which react with the DNA forming adducts and inter and intra-strand cross-links (Fleming, 1997; Hemminki, 1987; IARC Working Group on Male and female Swiss mice (7–8 weeks old, 25–30 g body weight) the Evaluation of Carcinogenic Risks to Humans, 2012). Liver phase II were housed under 12:12 h light:dark cycles, at standard temperature enzymes, such as glutathione transferase, associated with ABC trans- (20–25 °C) and humidity (60 ± 10%) conditions, with food and water porters like Mrp2 that dump their conjugated products into bile, are ad libitum. A total number of 64 animals were randomly divided into 8 essential for the elimination of CP and its metabolites in rat liver (Qiu groups per sex containing 4 males and 4 females per group in the an- et al., 2003). Interactions between ABC transporters and CP or its me- imal house facility at ININFA (Instituto de Investigaciones tabolites in other tissues have also been reported for BCRP, MRP4 and Farmacológicas – Universidad de Buenos Aires–CONICET). All proce- P-gp (Brayboy et al., 2013; Zhang et al., 2005). In addition, we have dures involving animals were conducted in accordance with the observed that D. tenuifolia intake modulates Bcrp as well as Mrp2 in guidelines for the Care and Use of Laboratory Animals (Committee for mouse liver (López Nigro et al., 2018). the Update of the Guide for the Care and Use of Laboratory Animals, Thus, the modulation of ABC transporters by natural product com- 2011) and Argentinian regulation for animal facilities (Administración pounds could affect the bioavailability of both CP and its active meta- Nacional de Medicamentos Alimentos y Tecnología Médica, 1996). bolites. The goal of the present study was to determine whether chronic intake of E. vesicaria has the ability to modulate hepatic ABC efflux transporters and the relevance of this interaction for its genotoxic or 2.3.1. Juice doses and animal treatment protective effect in vivo in mice as well as in vitro in human hepatic cells. E. vesicaria juice doses were selected on the basis of an approximate daily consumption of 70, 100 and 140 g of rocket salad for an average 2. Materials and methods adult, these being equivalent to one to two servings per day according to the guidelines (Hartmann et al., 2003; MacGregor et al., 1987). Male 2.1. Chemicals and female mice were treated according to Lopez Nigro et al. (López Nigro et al., 2018), as shown in Fig. 1. Briefly, animals were weighted Methanol (purity > 99%), Giemsa solution, modified May- Grünwald's eosine-methylene blue solution, NaCl (purity > 99%), di- methylsulfoxide (DMSO, purity > 99%) and NaOH (purity > 99%) were purchased from Merck (Argentina). Ethidium bromide (purity >

95%), acridine orange (purity > 95%), Na2EDTA (purity > 99%), Trizma Base (purity > 99%), Triton X-100, Tween 20, Cyclophosphamide, phenylmethanesulfonyl ﬂuoride (PMSF), sodium dodecyl sulphate (SDS), verapamil, (3S,6S,12aS)-1,2,3,4,6,7,12,12a- Octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6] pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester hydrate (Ko-143), indomethacin, trypan blue, 1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Fetal bovine serum, phosphate buﬀered saline, low- melting point agarose (LMPA) and regular agarose were purchased from Gibco (Waltham, Massachusetts, U.S.A.). Heparin was from Abbott (Illinois, U.S.A). Protease inhibitor cocktail (Complete mini) was Fig. 1. Treatment scheme. CP: cyclophosphamide, i.p.: intraperitoneal injec- purchased from Roche Applied Science (Mannheim, Germany). tion.

2 M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797 daily and a dose of 1.0, 1.4 or 2.0g/kg body weight of rocket juice for 2.5. In vivo assessment of the genotoxic and/or DNA protective properties of each treatment group were administered by gavage in a volume of Eruca vesicaria 0.01ml/g body weight for 14 consecutive days. Two groups re- presenting the negative and positive controls received a similar volume 2.5.1. Alkaline comet assay in mice blood cells dose of saline solution (0.9% NaCl). The comet assay (Single Cell Gel Electrophoresis) was performed in On day 15, half of the groups of mice received an intraperitoneal its alkaline version, following the protocol by Singh et al. with mod- injection (i.p.) of 50 mg/kg of CP (Positive Control and DNA Protection ifications (Singh et al., 1988). The animals were sacrificed by cervical groups), the other half received an i.p. injection of saline solution dislocation, and blood was rapidly extracted through cardiac puncture (Negative Control and Genotoxicity groups). On day 16, all mice were with a heparinized syringe. Great care was taken in order to avoid di- sacrificed by cervical dislocation to obtain heparinized blood, both fe- rect light exposure of the samples throughout the entire procedure. The mora and the liver. Mercille and Massie fluorescent method (Mercille and Massie, 1994), To evaluate the role of ABC transporter modulation on E. vesicaria which uses ethidium bromide and acridine orange to differentiate living antigenotoxicity, an additional group of 24 female mice was randomly and dead cells, was used to assess cell viability of the samples. Only divided into 6 groups of 4 animals each. Only females were used since samples that showed cell viability greater than 80% were considered no differences between genders were evidenced in neither genotoxicity adequate for this test. nor ABC transporter expression for the highest E. vesicaria juice dose. Microscope slides were coated with a layer of 1% regular agarose. All mice were weighted daily and received a dose of 2 g/kg body Afterwards, the blood samples were mixed with 0.5% low melting-point weight of E. vesicaria juice for 14 consecutive days. On day 15, they agarose at 37 °C and placed on top, with the addition of a final layer of received i.p. injections of: a) Group 2.0 g/kg + SS: saline solution; b) low melting-point agarose. These slides were immersed in cold lysing

Group 2.0 g/kg + CP: 50 mg/kg CP; c) Group 2.0 g/kg + V: 50 mg/kg solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 10% di- verapamil; d) Group 2.0 g/kg + V + CP: 50 mg/kg verapamil and methylsulfoxide and 1% Triton-X100, pH 10) and kept refrigerated 50 mg/kg CP; e) Group 2.0 g/kg + I: 10 mg/kg indomethacin; and f) overnight. Subsequently, DNA unwinding was carried out with cold

Group 2.0 g/kg + I + CP: 10 mg/kg indomethacin and 50 mg/kg CP. alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH Doses of ABC transporter inhibitors were selected on the basis of re- 13.5) for 20 min, generating single-strand DNA thus allowing the ex- ported evidences (Dahan and Amidon, 2010; Horton, 1989). pression of alkali-labile sites and single-strand breaks. Following this On day 16, mice were sacrificed by cervical dislocation and both step, electrophoresis was performed on a horizontal electrophoresis femora were collected. system (BioRad, Hercules, CA, USA) using electrophoresis buffer at a constant voltage of 0.7 V/cm and 300 mAmp for 20 min at 4 °C. Finally, the gels were neutralized with Tris 0.4 M, pH 7.5 and the slides were 2.4. Hepatic expression of ABC efflux transporters stained with 20 μg/ml ethidium bromide. The analysis was performed on an Olympus BX40 fluorescence microscope. For each sample, 100 2.4.1. Preparation of liver membranes comets in duplicate were scored and classified into four damage cate- Isolated livers from the negative and positive controls, and 1.0, 1.4, gories according to DNA migration, and a Damage Index (DI, in arbi- 2.0 g/kg E. vesicaria groups (n = 4 per group) were collected, rinsed in trary units) was calculated for each sample as follows: cold saline and promptly stored at −70 °C. Fraction enriched in plasma DI = n1+2xn2+3xn3+4xn4 (where nx represent the number of comets membrane was prepared by differential centrifugation after homo- in categories 1 to 4). In addition, Damage Reduction Indices (DRIs) genization in 0.3 M sucrose containing 0.1 mM phenylmethylsulfonyl were obtained (Serpeloni et al., 2008): fluoride, 25 mg/ml leupeptin, 5 mg/ml aprotinin and 5 mg/ml pep- Reduction (%)=×DI C+− DI X 100 whereC + represents the mean of DI C+− DI C − statin A (50 mg liver per ml of buffer) (López Nigro et al., 2018). the positive control group, X represents the genotoxicity observed in the mice that received E. vesicaria juice plus CP and C- represents the mean of the negative control group. 2.4.2. Western blot analysis Protein concentration in homogenates was measured (Lowry et al., 2.5.2. In vivo mice micronucleus test 1951) with BSA as standard. Proteins (30 μg/well) were separated on a The protocol for this assay was adapted from Schmid (1975). Both 7.5% SDS-polyacrylamide gel and transferred to a poly(vinylidene di- femuri were extracted and washed with saline solution. The epiphyses fluoride) (PVDF) membrane (Amersham Hybond GE Healthcare Life were cut, and the medullar canal was washed with a syringe containing Sciences, UK). The membranes were blocked with 5% nonfat milk fetal bovine serum and a 25G needle. The bone marrow cell suspension prepared in TBS-Tween 20 and were afterwards cut at molecular mass obtained was centrifuged and resuspended in fetal bovine serum. Slides 52 and 102 kDa, based on Full-Range Rainbow Molecular Weight were prepared by cytocentrifugation, air-dried and fixed with me- Markers (GE Healthcare Life Sciences, UK). For detection of ABC thanol. May-Grünwald/Giemsa staining was performed for the identi- transporter proteins, each portion of membrane was incubated with fication of polychromatic (PCE) and normochromatic (NCE) ery- primary antibodies as follows: mouse monoclonal anti MDR1 (anti P-gp; throcytes by their differential coloration. Five hundred erythrocytes ABCB1; D-11; IgG2b; 1:500; Santa Cruz Biotechnologies; CA, USA), were scored in order to obtain the Cytotoxicity Index: PCE/ mouse monoclonal anti ABCG2 (anti Bcrp, BXP-21; IgG2a; 1:800; Santa (PCE + NCE), and 1000 PCE and NCE were scored in duplicate for the Cruz Biotechnologies; CA, USA) and mouse monoclonal anti ABCC2 assessment of the frequency of micronuclei in 2000 PCE and NCE for (anti Mrp2; M2III-5; IgG2b; 1:150; Abcam; Cambridge, UK), diluted in each sample. A Reduction Index was determined (Serpeloni et al., 2008) 1% serum albumin in TBS-Tween 20. As load control, rabbit polyclonal in an analogous way to the comet assay: anti-actin (1:1000; Sigma-Aldrich) was used. Goat anti-rabbit horse- MicronucleiPCE C+− MicronucleiPCE X radish peroxidase-conjugated immunoglobulin G (sc-2004; 1:2000; Reduction (%)= × 100 MicronucleiPCE C+− MicronucleiPCE C − Santa Cruz Biotechnologies) and goat anti-mouse horseradish peroxidase-conjugated immunoglobulin G (sc-2005; 1:2000; Santa Cruz Biotechnologies) were used as secondary antibodies, respectively. 2.6. In vitro cell viability assays in human liver cells Chemiluminescence reaction (Amersham ECL Biosciences, Little Chal- font, UK) was used to visualize bands and optical densitometry was 2.6.1. Cell culture performed with NIH ImageJ free software. Assays were carried out in Human hepatic carcinoma cells C3A [HepG2/C3A, derivative of duplicate. HepG2 (ATCC HB¬8065)] (ATCC® CRL¬10741™) were kindly provided

3 M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797 by Dr. María Fernanda Troncoso (Espelt et al., 2019). Cells were cul- 3.2. Effect of chronic intake of E. vesicaria leaves juice on hepatic tured in Dulbecco's MEM medium supplemented with 10% fetal bovine expression of ABC transporters serum, 1% penicillin-streptomycin at 37 °C and 5% CO2 in a humidified incubator. Animal average weight gain as well as food and water consumption showed no significant differences between all the groups at the end of the treatment (p > 0.05). 2.6.2. Cell treatment with E. vesicaria juice, CP and ABC inhibitors As shown in Fig. 2, in control conditions, the protein expression of Cells were seeded in DMEM complete medium on 48 or 96-well P-gp (Fig. 2A) and Mrp2 (Fig. 2B) was significantly higher in females microplates (Greiner Bio-One) and cultured for 24 h. A concentration- compared to males (p < 0.05 and p < 0.01, respectively) whereas no response curve to CP (1–25 mM) showed that 10 mM CP caused a 50% sex-related differences were observed for the hepatic expression of Bcrp decrease in cell viability for the MTT assay as well as the Trypan Blue (Fig. 2C; p > 0.05). Moreover, no modifications on the expression exclusion test after 24 h. Following pretreatment for 48 h with profile of hepatic ABC transporters were found after 24 h of a single E.vesicaria (1.4 mg/ml) or its vehicle (saline solution), cells were further administration of CP (50 mg/kg, i.p.; p > 0.05), as shown in Fig. 2. exposed to 10 mM CP for 24 h alone or in the presence of verapamil With respect to the animal groups that received E. vesicaria juice, a (35 μM), KO-143 (10 μM, BCRP inhibitor) or indomethacin (80 μM). significant increase in protein abundance of hepatic P-gp was observed Culture medium was then replaced by fresh complete DMEM and cells in male (Fig. 3A) and female (Fig. 3D) mice treated with 1.4 mg/kg were cultured for further 24 h. (p < 0.05) and 2.0 mg/kg (p < 0.01), remaining unchanged for the lowest dose (p > 0.05). Additionally, a significant increase in Mrp2 was observed only for the highest dose (2.0 g/kg) in livers from both 2.6.3. MTT assay male and female mice (Fig. 3B and E; p < 0.05). On the other hand, no HepG2/C3A cells were seeded on 96-well microplates and treated as changes were observed on the expression of Bcrp for either sex (Fig. 3C described in section 2.6.2. Once the treatment was completed, medium and F; p > 0.05). was removed, and cells were washed with PBS. Fresh medium containing 0.45 mg/ml MTT was added to each well and cells were in- 3.3. Genotoxicity and DNA protective effect of E. vesicaria leaves juice cubated for 3 more hours for MTT reduction by living cells. Formazan crystals thus formed were dissolved in 100 μl of isopropanol at acid pH. No significant differences on genotoxicity biomarkers among male Absorbance at 570 nm and 660 nm (as a reference) were measured with and female mice from the same treatment group (p < 0.05) were a microplate spectrophotometer (FlexStation 3 Multi-Mode Microplate found, hence data from both genders were pooled. Reader). Results are expressed as the mean percentage of cell viability The comet assay showed that the DNA damage (expressed by the of four independent experiments. Damage Index in arbitrary units) of mice that received daily doses of E. vesicaria juice during 14 days was not significantly different from those receiving saline solution (Fig. 4A; p > 0.05). On the contrary, mice 2.6.4. Trypan blue exclusion test that received the rocket juice treatment and afterwards a CP injection HepG2/C3A cells were seeded in 48 well-plates and treated as de- evidenced significantly lower DNA damage than those that did not re- scribed in 2.6.2. After trypsinization, cells were diluted in the same ceived E.vesicaria juice (Fig. 4A; p < 0.001) for the three tested doses. volume of 0.5% trypan blue dye and the percentage of viable cells was Accordingly, the frequency of Mn in bone marrow PCE was not recorded by observation with a Neubauer hemocytometer on an optical different between saline solution and E. vesicaria juice-treated mice microscope (Nikon Eclipse) equipped with fluorescent optics. Results (p > 0.05). Nonetheless, there was a significant reduction in PCE MN were expressed as the mean percentage of cell viability of four in- frequency in mice that received the 3 doses of the juice (1.0, 1.4 and dependent experiments. 2.0 g/kg) and subsequently the CP injection in contrast with those that did not receive the juice treatment (p < 0.05, Fig. 4B). Furthermore, the Cytotoxicity Index (CI) allowed us to assess a 2.7. Statistical analysis potential cytotoxic effect which may interfere with the maturation ki- netics of the red blood cells precursors. No differences were observed Experimental data is expressed as mean ± SD and the statistical when the CI was compared between all the groups tested (Table 2; analysis was performed using IBM's SPSS software. Genotoxicity and p > 0.05). cell viability assays were analyzed by one way ANOVA, Kruskal-Wallis, Additionally, the frequency of micronuclei in NCE was recorded to post-hoc Dunnett's test for multiple comparisons, t-test and Mann- test genotoxicity prior to the administration of the DNA damage inducer Whitney's U test. Immunoblotting analysis was performed using CP (basal genotoxicity). All the groups evidenced frequencies compar- Kruskal-Wallis and post-hoc Dunn's test for multiple comparisons. able to those of negative controls (p > 0.05), suggesting that all mice had undergone no previous genotoxic exposures (Table 2). The Damage Reduction Indices (DRIs) for the comet assay and the 3. Results Mn test calculated for 1.0, 1.4 and 2.0 g/kg E. vesicaria juice were the following: a) for the comet assay: 61.80%, 72.76% and 79.27%; b) for 3.1. Quantitative phytochemical screening of Eruca vesicaria leaves juice the Mn test: 9.45%, 35.32% and 43.28%. Oral administration of the cruciferous was associated with a dose-dependent increase in DRIs for The results of the quantitative analysis of E. vesicaria juice (2.55 g/ both DNA damage biomarkers. Moreover, the E. vesicaria-induced in- ml) are presented in Table 1. crease in DRIs was higher for the comet assay than for the Mn test at each tested dose. Table 1 Quantitative phytochemical analysis of E. vesicaria juice (2.55 g/ml). 3.4. Effects of in vivo inhibition of ABC transporters on E. vesicaria Concentration (mean ± SD) antigenotoxicity

μ Total Flavonoids ( g rutin/mL) 65.82 ± 1.35 Following the same treatment scheme, we analyzed the role of ABC Hydroxycinnamic acid (μg chlorogenic acid/mL) 310.69 ± 10.88 ﬀ Total Phenols (μg gallic acid/mL) 526.0 ± 21.43 transporters in the antigenotoxic e ect of 2.0 g/kg E.vesicaria, which causes hepatic overexpression of P-gp and Mrp2 (see section 3.2.). The

4 M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797

Fig. 2. Effect of a single dose of CP on hepatic ABC transporter expression. Representative immunoblots of P-gp (A), Mrp2 (B), and Bcrp (C) in liver of adult male and female mice. Animals treated with saline solution during 14 days received an i.p. injection of saline solution (Negative control, light gray boxes) or 50 mg/kg body weight (dark gray boxes) on day 15. Densitometric units were calculated as ratio between optical density of the ABC transporter and the load control band. Results are expressed as median and the lower and upper quartiles of four mice per group. *p < 0.05 and **p < 0.01 between male and female for P-gp and Mrp2 expression, respectively, in the corresponding treatment (Kruskall Wallis with Dunn's post-hoc test). mice micronucleus test was selected for genotoxicity assessment due to 4. Discussion its robustness and the correlation between this biomarker and the comet assay that we observed in the first group of mice (Fig. 4A and B). The present study showed that repeated oral administration of fresh As shown in Fig. 5,nodifferences in the frequency of Mn in PCE leaves of E. vesicaria has a protective effect against genotoxicity caused were found between the group of animals that received only E. vesicaria by CP. This effect would depend, at least in part, on the overexpression juice and those that also received the P-gp inhibitor (50 mg/kg ver- of the hepatic efflux transporters P-gp and Mrp2 caused by sustained apamil; p > 0.05) or the Mrp2 inhibitor (10 mg/kg indomethacin; exposure to this cruciferous in mice. Moreover, an anti-cytotoxic effect p > 0.05). In accordance with results presented in section 3.3, mice of E.vesicaria dependent on P-gp, MRP2 and BCRP was also observed in that received CP following the juice treatment showed a significant vitro in human liver cells. increase of this biomarker (Fig. 5; p < 0.05). The alkaline comet assay in blood lymphocytes and the Mn test in Interestingly, mice that received E. vesicaria juice, CP and verapamil mice bone marrow are genotoxicity effect biomarkers frequently used or indomethacin evidenced a marked increase in the frequency of Mn, in a complementary way to detect different types of DNA damage in contrast with those that were exposed to the juice and CP only (Bowen et al., 2011; Kang et al., 2013). The comet assay, or single-cell (Fig. 5; p < 0.001). gel electrophoresis, detects DNA-strand breaks in virtually any type of Regarding CI and the frequency of Mn in NCE, no differences were cells including blood cells. The absence of modifications in the DNA found among any of the groups (p > 0.05, data not shown). damage index (DI) for the comet assay after the repeated ingestion of E. vesicaria leaf juice, showed that the crucifer did not cause any breaks or alkali-labile sites in the DNA of mice leucocytes. Regarding the in vivo 3.5. Effect of hepatic ABC transporter inhibition on the protective action of Mn test, during erythropoiesis, lagging chromosomes or acentric frag- E. vesicaria in human hepatocytes ments in the cytoplasm of dividing red blood cells precursors give rise to smaller nuclei after telophase, which are known as micronuclei Finally, to further analyze the involvement of hepatic ABC trans- (MacGregor et al., 1987; Schmid, 1975). These remain in the cytoplasm porters in the protective effect of E. vesicaria,anin vitro experimental of polychromatic erythrocytes, which are formed once the erythroblast set was carried out in human hepatocellular carcinoma cells, which expels its main nucleus. The fact that the MN frequencies were not display xenobiotic metabolism and ABC transporter pathways (HepG2/ increased in mice treated with E. vesicaria juice suggests that this cru- C3A). Cell viability was examined by two complementary assays: MTT ciferous is not clastogenic or aneugenic at the doses used. Additionally, assay (Fig. 6A) and Trypan Blue exclusion test (Fig. 6B). Selected only micronuclei in PCE are originated from a recent genotoxic insult, concentrations of ABC inhibitors were not cytotoxic per sé. As shown in whereas micronuclei in NCE are indicative of previous damage Fig. 6A and B, exposure to 1.4 mg/ml E. vesicaria juice did not affect cell (Mavournin et al., 1990; Schmid, 1975). According to this, we could viability. Nevertheless, 10 mM CP for 24 h produced a decrease of ap- corroborate that erythropoietic cells suffered no cytotoxic damage since proximately 50% in cell viability respect to the negative control, but PCE and NCE ratio (Cytotoxicity Index) was comparable among all pretreatment with E. vesicaria juice significantly reduced CP-induced groups. All together, these results show that repeated oral intake of E. cytotoxicity (Fig. 6A and B; p < 0.05). However, the protective effect vesicaria was not genotoxic in doses comparable to human average in- of E. vesicaria juice was prevented when the P-gp inhibitor (35 μM take. verapamil; Fig. 6A and B; p < 0.001) was co administered with CP. Cyclophosphamide toxicity is based on the high reactivity of the Similarly, coadministration of CP with the MRP2 inhibitor (80 μM in- nitrogen mustard ―one of its metabolites, generated through cyto- domethacin; Fig. 6A and B; p < 0.001) significantly reverted the pro- chrome P450― which interacts with guanine residues forming inter tective effect of the cruciferous. Interestingly, the BCRP inhibitor and intrastrand cross-links. This process leads to inhibition of DNA (10 μM Ko-143), also avoided the protective effect of E.vesicaria leaves synthesis and cytotoxicity (Baumann and Preiss, 2001). These char- juice on CP-induced cytotoxicity (Fig. 6A and B; p < 0.01). acteristics made cyclophosphamide an extremely suitable agent to be

5 M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797

Fig. 3. Effect of E. vesicaria juice on hepatic ABC transporter expression. Representative immunoblot of P-gp (A, D), Mrp2 (B, E), and Bcrp (C, F) in liver of adult male (A,B,C) or female (D, E, F) mice. Animals were treated with saline solution (white bars) or 1 g/kg (light gray bars), 1.4 g/kg (medium gray bars) or 2 g/kg (dark gray bars) of E. vesicaria juice. Densitometric units were calculated as ratio between optical density of transporter and the load control band. Bars represent protein levels as percentages of the average value from control group. Results are expressed as median and the lower and upper quartiles of four mice per group. * = p < 0.05 and ** = p < 0.01 versus negative control, respectively (Kruskall Wallis and Dunn's post-hoc test). used as a positive control in in vivo genotoxicity assessments (Krishna showing a significant inverse correlation between damage and juice et al., 1995; Organization for Economic Cooperation and Development dose (Pearson's R = −0.613, p < 0.001). For a deeper characteriza- (OECD), 2016). For that purpose, we treated three groups of mice with tion of this effect, a Damage Reduction Index was calculated for each an i.p. injection of 50 mg/kg of CP after chronic administration of the dose (refer to section 2.5) that confirmed a reduction of nearly eighty three doses of the vegetable juice (“protective effect” groups). The percent of the damage for the highest juice dose. comet assay revealed a significant decrease in DI for the three doses, Moreover, the significant decrease in the frequency of Mn in PCE for

Fig. 4. Genotoxicity biomarkers. A: DNA damage in mice blood cells.Bars represent mean ± SD of the damage index in arbitrary units obtained through the alkaline comet assay (8 animals per group); *p < 0.001 versus positive control (Kruskal-Wallis with Dunn's post- hoc test). B: Frequency of micronucleated polychromatic erythrocytes (PCE) of mice bone marrow. Bars represent mean ± SD of the number of micronuclei in2000 PCE (8 animals per group); *p < 0.05 versus positive control (one-way ANOVA with Dunnett's post-hoc test).

6 M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797

Table 2 eﬀect and juice dose (Spearman's R = −0.810, p < 0.001) allowed us Cytotoxicity Indices and frequencies of Mn in NCE. to conclude that the protective eﬀect of E. vesicaria was dose-depen-

Treatment CI (X ± SD) Mn/2000 NCE (X ± SD) dent. In addition, Damage Reduction Indices for the Mn test were lower than for the comet assay but exceeded forty percent for the highest Negative Control 0.52 ± 0.06 0.75 ± 0.71 dose. Positive Control 0.50 ± 0.08 1.00 ± 0.76 Since the health promoting effects of cruciferous vegetables has 1 g/kg b.w. + SS 0.55 ± 0.05 0.38 ± 0.74 1.4 g/kg b.w. + SS 0.54 ± 0.03 0.38 ± 0.74 been vastly attributed to glucosinolate content, we requested colla- 2 g/kg b.w. + SS 0.48 ± 0.06 0.63 ± 0.52 boration from Dr. Niels Agerbirk (Department of Plant and 1 g/kg b.w. + CP 0.55 ± 0.06 1.13 ± 1.13 Environmental Sciences, University of Copenhagen, Denmark) who 1.4 g/kg b.w. + CP 0.53 ± 0.06 1.13 ± 0.35 performed LC-MS for the detection of glucosinolates on our E. vesicaria 2 g/kg b.w. + CP 0.47 ± 0.06 0.63 ± 0.74 leaves. The main glucosinolates found in our accessions were glucor- SS: saline solution, CP: cyclophosphamide 50 mg/kg body weight. aphanin, glucoerucin, methoxyglucobrassicin and diglucothiobeinin (Bell et al., 2015; Lelario et al., 2012). However, these phytochemicals were present in extremely low concentrations below the limits of quantification of the method. Similar results were found in commercial varieties of E. vesicaria showing enhanced flavonol concentrations but extremely low concentration of glucosinolates (Bell et al., 2015). Nonetheless, we could identify and quantify other types of phytochemicals in our plant, these being flavonoids, phenols and hydroxycinnamic acids. Given the fact that they were present in much higher concentrations than glucosinolates (Table 1), we propose that these phytochemicals could be responsible for the antigenotoxic effects that we observed. Regarding the expression of ABC transporters, the significantly higher protein abundance of P-gp and Mrp2 in livers isolated from females compared to males in our experiments agrees with previous evidence reported in rodents proposing that these two transporters are regulated by sex hormones (Lu and Klaassen, 2007; Suzuki et al., 2006). Fig. 5. Effect of hepatic ABC transporter inhibition on E. vesicaria juice In turn, expression levels of hepatic ABC transporters remained antigenotoxicity. Frequency of micronucleated polychromatic erythrocytes unchanged after 24 h of a single i.p. dose of 50 mg/kg CP (Fig. 2). On (PCE) of mice bone marrow. Female mice were treated with 2.0 g/kg body fi weight of E. vesicaria juice. On day 15, animals were divided into six groups the other hand, a signi cant induction of the protein expression of P-gp receiving an i.p. injection of saline solution (SS) or 50 mg/kg cyclopho- and Mrp2 after chronic oral administration of E. vesicaria juice was sphamide (CP) alone or in addition to an i.p. injection of either the P-gp in- observed in both sexes (Fig. 3). This modulating effect of E. vesicaria hibitor verapamil (50 mg/kg; V) or the Mrp2 inhibitor indomethacin (10 mg/ could be attributed to flavonoids, as kaempferol interacts with Mrp2 kg; I). Two groups of animals were treated with verapamil or indomethacin and Bcrp transporters in vivo (Zheng et al., 2016). It is of relevance that alone for evaluation of basal toxicity of the ABC inhibitors. Bars represent the quali-quantitative phytochemical composition, and consequently mean ± SD of the number of micronuclei every 2000 PCE (4 animals per their actions on the organisms, differ between cruciferous species. In group); #p < 0.05 versus SS; **p < 0.001 versus CP (one-way ANOVA with this sense, Diplotaxis tenuifolia (wild rocket) shows a different pattern of Dunnett's post-hoc test). modulation of hepatic ABC transporters (López Nigro et al., 2018)in contrast with E. vesicaria (present results). Exposure to Diplotaxis te- the three juice doses of E. vesicaria suggests that this cruciferous also nuifolia do not induce hepatic expression of P-gp, but expression of exerted a protective effect against clastogenic damage induced by CP Mrp2 is decreased in females and increased in males showing sex-re- metabolites. Furthermore, the significant correlation between this lated differences; regarding Bcrp, its expression is strongly induced in

Fig. 6. Cytotoxicity assays in human liver cells. Bars represent percentage of viable HepG2/C3A cells respect to the negative control evaluated by MTT assay (Fig. 6A) or the Trypan Blue exclusion assay (Fig. 6B). Negative Control: saline solution; Positive control: 80 μM doxorubicin for 24 h; CP: 10 mM CP for 24 h; V: 35 μM verapamil for 24 h; K: 10 μM KO-143 for 24 h; I: 80 μM indomethacin for 24 h; 1.4 mg/ml: pretreatment with 1.4 mg/ml E. vesicaria juice for 48 h. Data represent the mean ± SD of four independent experiments. #p < 0.05 versus Negative control; ##p < 0.01 versus negative Control; ###p < 0.001 versus Negative control; a = *p < 0.05 versus CP; b = **p < 0.01 versus 1.4 mg/ml + CP, ***p < 0.001 versus 1.4 mg/ml + CP (Kruskal-Wallis, post-hoc Dunnett's test for multiple comparisons, t-test and Mann-Whitney's U test).

7 M.I. Roma, et al. Food and Chemical Toxicology 133 (2019) 110797 both male and female mice. Concerning participation of glucosinolates, possible that phytochemicals present in Eruca spp. act synergistically by although being present in very low concentrations in our accessions, it modulation of both systems, generating an antigenotoxic effect at doses could not be discarded for E. vesicaria since sulforaphane and erucine comparable to the average intake in humans. Repeated rocket intake induce P-gp and Mrp2 in human hepatic carcinoma cells (Harris and would facilitate the homeostatic response of the body in its protection Jeffery, 2008). The differential behavior of both species may lay within against environmental insults such as those elicited by oral exposure to variations in their phytochemical quantitative composition, since D. xenobiotics. tenuifolia contains significantly higher concentrations of total phenols Further in silico and in vitro experiments conducting to identify the and hydroxycinnamic acids (López Nigro et al., 2018 and Table 1 of this secondary metabolites involved in the modulation of ABC transporters manuscript). In this sense, consistent variations in the phenolic com- in each species are the goal of our future investigations. pounds between E. vesicaria and D. tenuifolia have been reported (Bell et al., 2015). These differences in the modulation pattern of hepatic 5. Conclusions ABC transporters could account for the differences found in the antigenotoxic effect of both cruciferous vegetables. Accordingly, the da- It appears that incorporation of fresh leaves of E. vesicaria to the diet mage reduction index (DRI) for both genotoxicity biomarkers is higher could induce the expression of hepatic ABC transporters contributing to for wild rocket than for common rocket (López Nigro et al., 2018 and reduce the risks of cellular burden of toxic compound exposure. These present results, respectively). results give further support to the beneficial effect that was previously Moreover, since ABC transporters are able to expel CP and its me- observed in vitro and in non-vertebrate species as Drosophyla melano- tabolites to the cellular exterior (Brayboy et al., 2017, 2013; Joy et al., ganster (Barillari et al., 2005; Villatoro-Pulido et al., 2012). 2012), the fact that repeated exposure to E. vesicaria positively modu- On the other hand, it should be taken into account that rocket lated the ABC transporters in liver could contribute to reduce CP-in- consumption could lead to drug-diet interactions that might negatively duced genotoxicity. Regarding Mrp2, it regulates the rate of biliary affect the treatment with agents that induce DNA damage, and with excretion of GSCP (metabolite of 4OH-CP). Although this metabolite is drugs whose pharmacokinetics depend on the action of ABC transpor- inactive, its higher accumulation in Mrp2-deficient rats facilitates the ters. oxidative metabolic pathway of 4OH-CP with the consequent increase in cytotoxicity (Qiu et al., 2003). In addition, a polymorphic variant of Funding P-gp, which correlates with altered expression and activity of the transporter, reduces the elimination rate of CP and 4OH-CP in patients This work was supported by the Universidad de Buenos Aires [grant with glomerulonephritis (Joy et al., 2012), thus indicating that P-gp is number UBACYT20920160100154BA, 2014–2017 and also involved in the pharmacokinetics of CP and its metabolites. In UBACYT20020170100637BA, 2018–2020] and by CONICET support of our hypothesis that ABC transporters participate in the an- [PIP11220120100499]. tigenotoxic action of E. vesicaria, the present findings show that the protective effect of the cruciferous against CP-induced genotoxicity is Declaration of competing interest reduced by P-gp (50 mg/kg verapamil) as well as Mrp2 (10 mg/kg indomethacin) inhibition (Fig. 5). On the light of this evidence, we pro- The authors declare that they have no known financial interests or pose that induction of hepatic Mrp2 and P-gp after chronic intake of E. personal relationships that could have appeared to influence the work vesicaria juice could facilitate biliary excretion of CP or its metabolites, reported in this paper. thus interfering with its genotoxicity in blood and bone marrow cells. To gain further knowledge on whether hepatic ABC transporters are Acknowledgements involved in the effect of E. vesicaria on CP-induced toxicity, we designed in vitro experiments in human hepatoma cells (HepG2/C3A). This cell The authors wish to thank Dr. Niels Agerbirk for the evaluation of line is a sub clone derived from HepG2 used as model for toxicological the glucosinolate profile of the E. vesicaria leaves used in this study at studies since it displays specialized functions of the normal liver such as the Department of Plant and Environmental Sciences, University of xenobiotic metabolism and ABC transporter pathways (Belkahla et al., Copenhagen, Denmark. Moreover, our thanks to Dr. María Fernanda 2018; Hewitt and Hewitt, 2004). Cytotoxic concentrations of CP Troncoso from Instituto de Química y Fisicoquímica Biológicas (10 mM) were evaluated by MTT and Trypan Blue assays, which evi- (IQUIFIB), Departamento de Química Biológica, Facultad de Farmacia y dence mitochondrial activity and cell membrane integrity, respectively. Bioquímica, Buenos Aires, Argentina, for providing HepG2/C3A cells. Under these experimental conditions, 1.4 mg/ml E. vesicaria juice at- tenuated CP-induced cytotoxicity on HepG2/C3A cells, but this effect References was no longer evident when P-gp (35 μM verapamil) or Mrp2 (80 μM indomethacin) were inhibited. Interestingly, in the human hepatoma Ebert, B., Seidel, A., Lampen, A., 2006. Phytochemicals induce breast cancer resistance cell line, BCRP was also involved in the protective effect of E. vesicaria. protein in caco-2 cells and enhance the transport of benzo[a]pyrene-3-sulfate. – fl fi Toxicol. Sci. 96, 227 236. https://doi.org/10.1093/toxsci/k 147. The discrepancies with in vivo ndings may be due to the fact that not Administración Nacional de Medicamentos Alimentos y Tecnología Médica, 1996. all orally administered compounds reach enough plasma concentrations Reglamentación para bioterios de laboratorios elaboradores de especialidades med- to exert their effect and, in addition, there are no equivalences between icinales y/o de análisis para terceros DISPOSICIÓN A.N.M.A.T. N° 6344/96. Aszalos, A., 2008. Role of ATP-binding cassette (ABC) transporters in interactions be- the doses used in vivo with respect to the concentrations used in in vitro tween natural products and drugs. Curr. Drug Metabol. 9, 1010–1018. tests. However, possible differences between species cannot be ruled Baasanjav-Gerber, C., Monien, B.H., Mewis, I., Schreiner, M., Barillari, J., Iori, R., Glatt, out. H., 2011. Identification of glucosinolate congeners able to form DNA adducts and to – Present findings suggest that efflux of CP and/or its metabolites induce mutations upon activation by myrosinase. Mol. Nutr. Food Res. 55, 783 792. https://doi.org/10.1002/mnfr.201000352. through ABC transporters may diminish their toxicity, and an increase Baiceanu, E., Crisan, G., Loghin, F., Falson, P., 2015. Modulators of the human ABCC2: in their expression of these efflux pumps could be one of the mechan- hope from natural sources? Future Med. Chem. 7, 2041–2063. https://doi.org/10. isms through which E. vesicaria exerts its protective effect. Furthermore, 4155/fmc.15.131. Barillari, J., Canistro, D., Paolini, M., Ferroni, F., Pedulli, G.F., Iori, R., Valgimigli, L., they add evidence to that already reported by Lamy, who observed that 2005. 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